Intracellular Staining by Flow Cytometry
|Detection of IL‑22 in Human PBMCs stimulated to induce Th17 Cells by Flow Cytometry. CD4+ Human peripheral blood mononuclear cells (PBMCs) either (A) stimulated to induce Th17 cells with 10 ug/mL Mouse Anti-Human CD3 epsilon Monoclonal Antibody (Catalog # MAB100), 5 ug/mL Mouse Anti-Human CD28 Monoclonal Antibody (Catalog # MAB342), 10 ng/mL Recombinant Human TGF‑ beta 1 (Catalog # 240-B), 20 ng/mL Recombinant Human IL‑2 (Catalog # 202-IL), 20 ng/mL Recombinant Human IL‑23 (Catalog # 1290-IL), 40 ng/mL Recombinant Human IL‑6 (Catalog # 206-IL), and 10 ng/mL Recombinant Human IL‑1 beta /IL‑1F2 (Catalog # 201-LB) for 5 to 7 days, then re-stimulated with 50 ng/mL PMA and 200 ng/mL Calcium ionomycin for 2 to 4 hours or (B) resting CD4+ PBMCs were stained with Mouse Anti-Human CD4 PE‑conjugated Monoclonal Antibody (Catalog # FAB3791P) and Mouse Anti-Human IL‑22 APC‑conjugated Monoclonal Antibody (Catalog # IC7821A). Quadrant markers were set based on isotype control (Catalog # IC002A). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin. View our protocol for Staining Intracellular Molecules.|
Interleukin-22 (IL-22), also known as IL-10-related T cell-derived inducible factor (IL-TIF) was initially identified as a gene induced by IL-9 in mouse T cells and mast cells. Human IL-22 cDNA encodes a 179 amino acid (aa) residue protein with a putative 33 aa signal peptide that is cleaved to generate a 147 aa mature protein that shares approximately 79% and 22% aa sequence identity with mouse IL-22 and human IL-10, respectively. The human IL-22 gene is localized to chromosome 12q15. Although it exists as a single copy gene in human and in many mouse strains, the mouse IL-22 gene is duplicated in some mouse strains including C57B1/6, FVB and 129. The two mouse genes designated IL-TIF alpha and IL-TIF beta, share greater than 98% sequence homology in their coding region. IL-22 has been shown to activate STAT1 and STAT3 in several hepatoma cell lines and upregulate the production of acute phase proteins. IL-22 is produced by normal T cells upon anti-CD3 stimulation in humans. Mouse IL-22 expression is also induced in various organs upon lipopolysaccharide injection, suggesting that IL-22 may be involved in inflammatory responses. The functional IL-22 receptor complex consists of two receptor subunits, IL-22 R (previously an orphan receptor named CRF2-9) and IL-10R beta (previously known as CRF2-4), belonging to the class II cytokine receptor family.
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